The intestinal epithelium controls the interaction between intestinal lumen contents and host immune cells. Microbial and dietary macromolecules (>600Da) can be transported across the intestinal epithelium through absorptive cells (enterocytes). In healthy humans, this process occurs in small doses and promotes tolerance of immune cells to the microbial and dietary antigens perpetually present in the gut lumen. In contrast, increased enterocyte transcellular macromolecule transport (ETMT) is associated with inflammatory bowel diseases (IBD), celiac disease, and food allergies. Despite the significance of ETMT in digestive diseases, the molecular mechanisms that govern this process are relatively understudied. Although great strides have been made in understanding the distinguishing features of this process, many aspects of the specific molecular mechanisms that control ETMT still remain unknown. Interestingly, gnotobiotic rodent studies have shown that the commensal gut microbiota stimulates ETMT. However, the microbial signals and host signal transduction mechanisms underlying this response have not been defined. The long-term objectives of this research are to increase knowledge of the specific molecular events involved in ETMT and how the microbiota contributes to this process. Towards achieving these goals, the specific aims of this research proposal are to 1) elucidate the specific endocytic mechanisms of ETMT that are stimulated by the commensal microbiota, 2) identify the microbial factors that stimulate ETMT, and 3) define the host signaling pathways that mediate microbial stimulation of ETMT. One factor that has traditionally hindered ETMT research is the paucity of experimental systems that accurately replicate the complex physiology of the gut environment and allow for real-time in vivo monitoring of absorptive processes. In these studies, a novel gnotobiotic zebrafish model will be utilized to identify the microbial signals and host response pathways that result in enhanced ETMT in intestinal enterocytes. The expected outcomes of the proposed research include unprecedented insights into the dynamic process of ETMT in a living vertebrate intestine, and the mechanisms utilized by enteric microbes to enhance ETMT. These results are expected to have a positive impact because they will lead to new probiotic and pharmacologic strategies for treating celiac disease, food allergy, and IBD by specifically limiting ETMT.